2 MATERIALS AND METHODS
In 2021, a clinical (lung) sample, TZJ2134, was collected in Shandong Province of China from a pig with suspected PRRSV infection. Tissue sample processing, RNA extraction, cDNA preparation, RT–PCR and genome sequencing were performed as described previously (Leng et al., 2014; Zhang. et al., 2015). TZJ2134 was identified as PRRSV-1 by RT–PCR with the primer L12. Based on the complete genomic sequence of PRRSV-1, eight primer pairs were designed for RT–PCR amplification and sequencing (Table S2), and two overlapping fragments of TZJ2134 were amplified by RT–PCR. Each PCR product was purified with the E.Z.N.A.® Gel Extraction Kit - Omega Bio-Tek, cloned into pMD18-T according to the manufacturer’s instructions, and then submitted to Comate Bioscience Co., Ltd. (Changchun, China) for sequencing.
Sequence analysis was performed with DNASTAR (version 7.1) software. The resulting sequences were assembled using SeqMan. Phylogenetic trees and molecular evolutionary analyses were constructed by using the neighbor-joining method in MEGA 7.0. with 1000 bootstrap replicates for each node (Kumar, Stecher, & Tamura, 2016). The generated phylogenetic tree was annotated using iTOL online software (https://itol.embl.de/) (Letunic & Bork, 2021). Sixty-five strains of PRRSV-1 available in GenBank were used for comparative sequence analyses in this study.
To test for recombination, the obtained sequence was screened using recombination detection program 4 (RDP4) (Martin et al., 2015). Seven different algorithms (RDP, GeneConv, BootScan, MaxChi, Chimera, SiScan, and 3Seq) embedded in the RDP4 software package with Bonferroni correction were utilized to detect recombination events and breakpoints. Detection using four or more of the seven methods implemented in RDP4 was taken as significant evidence for recombination. Recombination events were further confirmed by SimPlot 3.5.1 (Lole et al., 1999), and boot scanning analysis was performed with a 200-bp window, sliding along the genome alignment with a step size of 20 bp. The other three strains (PRRS-FR-2014-56-11-1, DK-2011-05-23-9 and OLot/91 strain) with high homology to TZJ2134-(A+B) were analyzed by SimPlot 3.5.1 (Lole et al., 1999) using the same method.